Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.09.003
Jorge Martinez-Laso , Isabel Cervera , María José Muñoz-Gómez , Clara Sánchez-Menéndez , Ana Salamanca-Soto , Montserrat Torres , Mayte Coiras
TLR8 and TLR9 are innate immune receptors belonging to the TLR family that are essential for viral recognition and early immune activation. Their dysfunction is linked to increased susceptibility to infections. TLR8 detects viral single- and double-stranded RNA, while TLR9 recognizes viral DNA molecules with CpG motifs. Six TLR8 alternative transcripts (V1, V2, V5, V6, V7, and V8) and nine TLR9 (V1, A, B, C, D, E, V8, V9, and V10) have been previously described in humans. In the present study, we have performed a comprehensive analysis of TLR8 and TLR9 transcripts in a healthy population and two new TLR8 transcripts (V3 and V4) and four new TLR9 transcripts (V2, V5, V6, and V7) were found. The main mechanisms for the generation of different mRNA variants were the insertion of non-coding regions and the loss of whole or partial exons. These changes result in the loss or insertion of new amino acids but only modify the initial leucine-rich repeat (LRR) region and preserve the rest of the receptor's complete structure. From the results obtained, we can deduce that there seems to be a strong evolutionary drive to maintain TLR8 and TLR9 functionality, unlike other innate immune response receptors.
{"title":"Novel alternative transcripts of TLR8 and TLR9 reveal evolutionary pressure to conserve protein structure","authors":"Jorge Martinez-Laso , Isabel Cervera , María José Muñoz-Gómez , Clara Sánchez-Menéndez , Ana Salamanca-Soto , Montserrat Torres , Mayte Coiras","doi":"10.1016/j.biochi.2025.09.003","DOIUrl":"10.1016/j.biochi.2025.09.003","url":null,"abstract":"<div><div>TLR8 and TLR9 are innate immune receptors belonging to the TLR family that are essential for viral recognition and early immune activation. Their dysfunction is linked to increased susceptibility to infections. TLR8 detects viral single- and double-stranded RNA, while TLR9 recognizes viral DNA molecules with CpG motifs. Six TLR8 alternative transcripts (V1, V2, V5, V6, V7, and V8) and nine TLR9 (V1, A, B, C, D, E, V8, V9, and V10) have been previously described in humans. In the present study, we have performed a comprehensive analysis of TLR8 and TLR9 transcripts in a healthy population and two new TLR8 transcripts (V3 and V4) and four new TLR9 transcripts (V2, V5, V6, and V7) were found. The main mechanisms for the generation of different mRNA variants were the insertion of non-coding regions and the loss of whole or partial exons. These changes result in the loss or insertion of new amino acids but only modify the initial leucine-rich repeat (LRR) region and preserve the rest of the receptor's complete structure. From the results obtained, we can deduce that there seems to be a strong evolutionary drive to maintain TLR8 and TLR9 functionality, unlike other innate immune response receptors.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 261-264"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145016936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The rapid expansion of available three-dimensional protein structures—derived from both experimental techniques and bioinformatic predictions—offers unprecedented opportunities for drug discovery, particularly for targets that have historically been difficult to characterize. However, the effective analysis of these increasingly complex and voluminous structural datasets remains a major challenge. Efficient representations of protein conformations are essential to facilitate large-scale comparison, structural classification, and functional interpretation in therapeutic contexts. The concept of structural alphabets, introduced by Pr S. Hazout in 1999, provides a robust and scalable framework to represent local protein backbone conformations using a limited set of recurring structural motifs. This representation enables a one-dimensional encoding of three-dimensional protein structures that retains essential geometric features beyond secondary structure, while allowing systematic and interpretable analyses. In this review, we focus on HMM-SA, a structural alphabet constructed using a hidden Markov model. HMM-SA defines 27 structural motifs, including 18 regions specifically dedicated to loops, and captures the statistical dependencies between them. We present a detailed overview of the HMM-SA framework, and of the computational tools derived from this structural alphabet, developed to explore protein function, conformational variability, and the structural determinants of molecular recognition. The utility of HMM-SA is illustrated through a case study on HIV-2 protease (PR2), a critical enzyme in antiretroviral drug development. By analyzing PR2 structural asymmetry, ligand-induced conformational changes, and mutation-driven alterations, we highlight the ability of HMM-SA–based methods to identify key structural features involved in ligand specificity and resistance mechanisms, thereby advancing therapeutic target analysis.
可获得的三维蛋白质结构的快速扩展——来自实验技术和生物信息学预测——为药物发现提供了前所未有的机会，特别是对于历史上难以表征的靶标。然而，对这些日益复杂和庞大的结构数据集进行有效分析仍然是一个主要挑战。蛋白质构象的有效表征对于促进大规模比较、结构分类和治疗背景下的功能解释至关重要。结构字母的概念是由Pr S. Hazout在1999年提出的，它提供了一个强大的、可扩展的框架，使用一组有限的重复结构基序来表示局部蛋白质主链构象。这种表示使三维蛋白质结构的一维编码保留了二级结构以外的基本几何特征，同时允许系统和可解释的分析。在这篇综述中，我们关注HMM-SA，一个使用隐马尔可夫模型构建的结构字母表。HMM-SA定义了27个结构基元，包括18个专门用于循环的区域，并捕获了它们之间的统计依赖关系。我们详细概述了HMM-SA框架，以及从该结构字母表衍生的计算工具，这些工具用于探索蛋白质功能、构象变异性和分子识别的结构决定因素。通过HIV-2蛋白酶（PR2）的案例研究说明了HMM-SA的效用，PR2是抗逆转录病毒药物开发中的关键酶。通过分析PR2结构不对称、配体诱导的构象变化和突变驱动的改变，我们强调了基于hmm - sa的方法识别与配体特异性和耐药机制相关的关键结构特征的能力，从而推进了治疗靶点分析。
{"title":"Exploring therapeutic targets with the HMM-SA structural alphabet: Methods, tools, and application to HIV-2 protease","authors":"Anne-Claude Camproux, Marine Baillif, Léa Dufay, Leslie Regad","doi":"10.1016/j.biochi.2025.08.001","DOIUrl":"10.1016/j.biochi.2025.08.001","url":null,"abstract":"<div><div>The rapid expansion of available three-dimensional protein structures—derived from both experimental techniques and bioinformatic predictions—offers unprecedented opportunities for drug discovery, particularly for targets that have historically been difficult to characterize. However, the effective analysis of these increasingly complex and voluminous structural datasets remains a major challenge. Efficient representations of protein conformations are essential to facilitate large-scale comparison, structural classification, and functional interpretation in therapeutic contexts. The concept of structural alphabets, introduced by Pr S. Hazout in 1999, provides a robust and scalable framework to represent local protein backbone conformations using a limited set of recurring structural motifs. This representation enables a one-dimensional encoding of three-dimensional protein structures that retains essential geometric features beyond secondary structure, while allowing systematic and interpretable analyses. In this review, we focus on HMM-SA, a structural alphabet constructed using a hidden Markov model. HMM-SA defines 27 structural motifs, including 18 regions specifically dedicated to loops, and captures the statistical dependencies between them. We present a detailed overview of the HMM-SA framework, and of the computational tools derived from this structural alphabet, developed to explore protein function, conformational variability, and the structural determinants of molecular recognition. The utility of HMM-SA is illustrated through a case study on HIV-2 protease (PR2), a critical enzyme in antiretroviral drug development. By analyzing PR2 structural asymmetry, ligand-induced conformational changes, and mutation-driven alterations, we highlight the ability of HMM-SA–based methods to identify key structural features involved in ligand specificity and resistance mechanisms, thereby advancing therapeutic target analysis.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 39-57"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144818644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.09.009
Wen-Feng Hu , Juan-Juan Wang , Jie Yu, Jun-Jie Yao, Ya-Li Liu, Zeng-Guang Xu, Zhan-Yun Guo
C-X-C motif chemokine ligand 17 (CXCL17) functions as a chemoattractant, though its receptor has been controversial. Recent independent studies, including our own, identified CXCL17 as an agonist for the orphan G protein-coupled receptor 25 (GPR25). While GPR25 orthologs are found across fishes to mammals, CXCL17 orthologs appear to be mammalian-specific, leaving the endogenous ligand for non-mammalian GPR25 orthologs unknown. This study unexpectedly found that human CXCL17 exhibits high activity towards GPR25 orthologs from the zebrafish (Danio rerio) and coelacanth (Latimeria chalumnae). Recombinant human CXCL17 efficiently activated both fish GPR25 orthologs in a NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay, and induced chemotactic movement in transfected human embryonic kidney (HEK) 293T cells expressing fish GPR25. A human CXCL17 mutant lacking three C-terminal residues showed no such effect. A NanoBiT-based binding assay revealed that a SmBiT-tagged human CXCL17 C-terminal fragment specifically bound to secretory large NanoLuc fragment (sLgBiT)-fused fish GPR25 orthologs. Fish GPR25 orthologs had significantly higher cell surface expression in transfected HEK293T cells compared to human GPR25, improving β-arrestin recruitment assay data quality. Despite approximately 400 million years of divergence between humans and fishes, the high activity of human CXCL17 on fish GPR25 orthologs suggests that the CXCL17–GPR25 pair may be conserved across all vertebrates, even though non-mammalian CXCL17 orthologs remain unidentified.
{"title":"Human CXCL17 binds and activates fish GPR25 orthologs","authors":"Wen-Feng Hu , Juan-Juan Wang , Jie Yu, Jun-Jie Yao, Ya-Li Liu, Zeng-Guang Xu, Zhan-Yun Guo","doi":"10.1016/j.biochi.2025.09.009","DOIUrl":"10.1016/j.biochi.2025.09.009","url":null,"abstract":"<div><div>C-X-C motif chemokine ligand 17 (CXCL17) functions as a chemoattractant, though its receptor has been controversial. Recent independent studies, including our own, identified CXCL17 as an agonist for the orphan G protein-coupled receptor 25 (GPR25). While GPR25 orthologs are found across fishes to mammals, CXCL17 orthologs appear to be mammalian-specific, leaving the endogenous ligand for non-mammalian GPR25 orthologs unknown. This study unexpectedly found that human CXCL17 exhibits high activity towards GPR25 orthologs from the zebrafish (<em>Danio rerio</em>) and coelacanth (<em>Latimeria chalumnae</em>). Recombinant human CXCL17 efficiently activated both fish GPR25 orthologs in a NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay, and induced chemotactic movement in transfected human embryonic kidney (HEK) 293T cells expressing fish GPR25. A human CXCL17 mutant lacking three C-terminal residues showed no such effect. A NanoBiT-based binding assay revealed that a SmBiT-tagged human CXCL17 C-terminal fragment specifically bound to secretory large NanoLuc fragment (sLgBiT)-fused fish GPR25 orthologs. Fish GPR25 orthologs had significantly higher cell surface expression in transfected HEK293T cells compared to human GPR25, improving β-arrestin recruitment assay data quality. Despite approximately 400 million years of divergence between humans and fishes, the high activity of human CXCL17 on fish GPR25 orthologs suggests that the CXCL17–GPR25 pair may be conserved across all vertebrates, even though non-mammalian CXCL17 orthologs remain unidentified.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 244-251"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145093078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.07.013
Isabelle Callebaut, Jean-Paul Mornon
Hydrophobic Cluster Analysis (HCA) adds secondary structure information to the analysis of protein amino acid sequence. Focusing on the elementary building blocks of protein folds, this approach has proved to be a powerful tool for detecting distant (hidden) relationships between proteins. At a time when huge masses of data are now available, both in terms of protein sequences and models of three-dimensional structures, it still constitutes a relevant tool for analyzing structural features at the scale of whole proteomes, enabling, among other things, to characterize the continuum between disorder and order and to explore the characteristics of protein dark matter. The aim of this mini-review is to provide a brief overview of this approach, describing its principles and achievements, recent developments and future prospects.
{"title":"Hydrophobic cluster analysis at protein and proteome scales","authors":"Isabelle Callebaut, Jean-Paul Mornon","doi":"10.1016/j.biochi.2025.07.013","DOIUrl":"10.1016/j.biochi.2025.07.013","url":null,"abstract":"<div><div>Hydrophobic Cluster Analysis (HCA) adds secondary structure information to the analysis of protein amino acid sequence. Focusing on the elementary building blocks of protein folds, this approach has proved to be a powerful tool for detecting distant (hidden) relationships between proteins. At a time when huge masses of data are now available, both in terms of protein sequences and models of three-dimensional structures, it still constitutes a relevant tool for analyzing structural features at the scale of whole proteomes, enabling, among other things, to characterize the continuum between disorder and order and to explore the characteristics of protein dark matter. The aim of this mini-review is to provide a brief overview of this approach, describing its principles and achievements, recent developments and future prospects.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 27-31"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144621463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.08.007
Bernard Offmann , Alexandre G. de Brevern
Protein Blocks (PBs) represent a widely used structural alphabet that enables the approximation and analysis of local protein conformations through 16 prototype fragments defined by dihedral angles. Initially developed to overcome the limitations of classical secondary structure definitions, PBs provide a powerful tool for understanding protein structure, dynamics, and function. Their applications span structural annotation, protein fold superimposition and recognition, sequence-based prediction and molecular dynamics analysis. Notably, PBs facilitate the distinction between rigid, flexible, and disordered regions via an entropy-based index (Neq), offering insights into protein flexibility and intrinsic disorder. Their integration with deep learning has dramatically improved predictive performance, and their utility has been demonstrated in diverse contexts such as integrin polymorphisms, VHH variability and AlphaFold structure analysis. As a robust and adaptable framework, PBs remain central in modern structural bioinformatics.
{"title":"A 25-year journey with protein blocks: Unveiling the versatility of a structural alphabet","authors":"Bernard Offmann , Alexandre G. de Brevern","doi":"10.1016/j.biochi.2025.08.007","DOIUrl":"10.1016/j.biochi.2025.08.007","url":null,"abstract":"<div><div>Protein Blocks (PBs) represent a widely used structural alphabet that enables the approximation and analysis of local protein conformations through 16 prototype fragments defined by dihedral angles. Initially developed to overcome the limitations of classical secondary structure definitions, PBs provide a powerful tool for understanding protein structure, dynamics, and function. Their applications span structural annotation, protein fold superimposition and recognition, sequence-based prediction and molecular dynamics analysis. Notably, PBs facilitate the distinction between rigid, flexible, and disordered regions via an entropy-based index (<em>N</em><sub>eq</sub>), offering insights into protein flexibility and intrinsic disorder. Their integration with deep learning has dramatically improved predictive performance, and their utility has been demonstrated in diverse contexts such as integrin polymorphisms, V<sub>H</sub>H variability and AlphaFold structure analysis. As a robust and adaptable framework, PBs remain central in modern structural bioinformatics.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 58-71"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144823413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.08.013
Isabele G. Frasson-Uemura , Franciele P. Dall’Aqua , Lunna U. Bosquetti , Otávio Vinícius C. Jorge , Thais T. Braga , Maria F. Siqueira , Manoel O.E. Favaro , Vania G.M. Mattaraia , Rui Curi , Roberto B. Bazotte , Priscila Cassolla , Gisele Lopes Bertolini
Liver glycogen catabolism was investigated in young adult Goto-Kakizaki rats (GK group) and compared with non-diabetic Wistar rats (Control group). The diabetic condition of GK rats was confirmed by hyperglycemia and insulin resistance. Glycogen catabolism was intensified during the infusion of epinephrine (10 μM, 20 μM, and 40 μM), phenylephrine (2 μM, 4 μM, and 6 μM), and glucagon (1 nM) in both Control and GK livers. The degree of glycogen catabolism during these infusions was similar in Control and GK rats, despite the higher liver glycogen content observed in the GK rats. However, a diminished intensification of hepatic glucose production was observed in GK rats during the infusion of isoproterenol (10 μM, 20 μM, and 40 μM). To further investigate this difference, the effect of cAMP, the intracellular mediator of isoproterenol, on liver glycogen catabolism was examined. Livers from GK rats showed no response to 3 μM and 5 μM cAMP but displayed a similar intensification of glycogen catabolism at 7 μM and 9 μM cAMP as the Control group. Interestingly, a higher intensification of glycogen catabolism was observed in GK livers during the infusion of 3 μM dibutyryl-cAMP, a phosphodiesterase-resistant cAMP analog, suggesting that cAMP inactivation by phosphodiesterases might be increased in GK livers. While these findings suggest a possible involvement of phosphodiesterases in the reduced response to isoproterenol, the evidence is insufficient to conclusively establish this mechanism. It can be concluded that liver glycogenolysis does not contribute to the hyperglycemia and glucose intolerance observed in young adult GK rats and that cAMP-mediated intracellular signaling appears to be attenuated in the livers of these animals.
{"title":"Liver glycogen catabolism in young adult Goto-Kakizaki rats","authors":"Isabele G. Frasson-Uemura , Franciele P. Dall’Aqua , Lunna U. Bosquetti , Otávio Vinícius C. Jorge , Thais T. Braga , Maria F. Siqueira , Manoel O.E. Favaro , Vania G.M. Mattaraia , Rui Curi , Roberto B. Bazotte , Priscila Cassolla , Gisele Lopes Bertolini","doi":"10.1016/j.biochi.2025.08.013","DOIUrl":"10.1016/j.biochi.2025.08.013","url":null,"abstract":"<div><div>Liver glycogen catabolism was investigated in young adult Goto-Kakizaki rats (GK group) and compared with non-diabetic Wistar rats (Control group). The diabetic condition of GK rats was confirmed by hyperglycemia and insulin resistance. Glycogen catabolism was intensified during the infusion of epinephrine (10 μM, 20 μM, and 40 μM), phenylephrine (2 μM, 4 μM, and 6 μM), and glucagon (1 nM) in both Control and GK livers. The degree of glycogen catabolism during these infusions was similar in Control and GK rats, despite the higher liver glycogen content observed in the GK rats. However, a diminished intensification of hepatic glucose production was observed in GK rats during the infusion of isoproterenol (10 μM, 20 μM, and 40 μM). To further investigate this difference, the effect of cAMP, the intracellular mediator of isoproterenol, on liver glycogen catabolism was examined. Livers from GK rats showed no response to 3 μM and 5 μM cAMP but displayed a similar intensification of glycogen catabolism at 7 μM and 9 μM cAMP as the Control group. Interestingly, a higher intensification of glycogen catabolism was observed in GK livers during the infusion of 3 μM dibutyryl-cAMP, a phosphodiesterase-resistant cAMP analog, suggesting that cAMP inactivation by phosphodiesterases might be increased in GK livers. While these findings suggest a possible involvement of phosphodiesterases in the reduced response to isoproterenol, the evidence is insufficient to conclusively establish this mechanism. It can be concluded that liver glycogenolysis does not contribute to the hyperglycemia and glucose intolerance observed in young adult GK rats and that cAMP-mediated intracellular signaling appears to be attenuated in the livers of these animals.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 139-149"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144823415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. In Chlamydomonas reinhardtii, phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. Although prior studies have identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, its broader interaction network and regulatory mechanisms remain poorly understood. In this study, we identified novel protein partners of phototropin and their roles in modulating its regulatory functions in C. reinhardtii. Employing a range of intraflagellar transport (IFT) mutants of C. reinhardtii, we demonstrated that phototropin localization to the flagella and eyespot is IFT-mediated. Our results reveal novel interactions between phototropin and other photoreceptors, including-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and the signaling protein- 14-3-3. CRISPR-Cas9 generated knockouts of phototropin led to reduced expression of ChR1 and 14-3-3, accompanied by impaired photomotility of the mutants. Additionally, gene expression of LOV-HK1 and LOV-HK2 were found to be elevated under UV-light in C. reinhardtii and these had altered expression in phototropin knockout line. These findings provide novel insights into phototropin interactome and elucidate molecular mechanisms underlying its localization and signaling functions in C. reinhardtii. This work advances our understanding of phototropin-mediated signal transduction and lays the groundwork for future exploration of its broader physiological roles in cellular responses.
{"title":"Phototropin localization and interactions regulate photophysiological processes in Chlamydomonas reinhardtii","authors":"Sunita Sharma , Kumari Sushmita , Rajani Singh , Sibaji K. Sanyal , Suneel Kateriya","doi":"10.1016/j.biochi.2025.08.014","DOIUrl":"10.1016/j.biochi.2025.08.014","url":null,"abstract":"<div><div>Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. In <em>Chlamydomonas reinhardtii</em>, phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. Although prior studies have identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, its broader interaction network and regulatory mechanisms remain poorly understood. In this study, we identified novel protein partners of phototropin and their roles in modulating its regulatory functions in <em>C. reinhardtii</em>. Employing a range of intraflagellar transport (IFT) mutants of <em>C. reinhardtii</em>, we demonstrated that phototropin localization to the flagella and eyespot is IFT-mediated. Our results reveal novel interactions between phototropin and other photoreceptors, including-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and the signaling protein- 14-3-3. CRISPR-Cas9 generated knockouts of <em>phototropin</em> led to reduced expression of <em>ChR1</em> and <em>14-3-3</em>, accompanied by impaired photomotility of the mutants. Additionally, gene expression of <em>LOV-HK1</em> and <em>LOV-HK2</em> were found to be elevated under UV-light in <em>C. reinhardtii</em> and these had altered expression in <em>phototropin</em> knockout line. These findings provide novel insights into phototropin interactome and elucidate molecular mechanisms underlying its localization and signaling functions in <em>C. reinhardtii</em>. This work advances our understanding of phototropin-mediated signal transduction and lays the groundwork for future exploration of its broader physiological roles in cellular responses.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 150-162"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144877237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.09.002
Ting Kang , Ruizhe Huang , Ruiheng Wang , Han Liu , Siyu Chen
The nuclear factor of activated T cells 3 (NFATc3) plays a significant role in various cancer-related processes, but its interactions with transcriptional modulators, particularly Promyelocytic Leukemia protein (PML), remain poorly understood. PML, a nuclear scaffold protein, is involved in tumor suppression and transcriptional regulation. This study investigates the interaction between NFATc3 and PML, focusing on the role of SUMOylation and its impact on downstream target genes. In vitro experiments, including mass spectrometry and Co-immunoprecipitation (Co-IP), were conducted to explore this interaction. Additionally, constructs with lysine-to-arginine (K→R) mutations at key SUMOylation sites were generated to determine whether PML SUMOylation is necessary for its interaction with NFATc3. We also assessed the impact of NFATc3 SUMOylation on its binding to PML. Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR) were employed to measure the expression of downstream genes (Lgr5 and Olfm4) under NFATc3 and PML overexpression or knockdown conditions. Pharmacological treatment with arsenic sulfide (As4S4) was used to further investigate modulation of the PML-NFATc3 axis. Our findings revealed that the NFATc3-PML interaction is independent of the SUMOylation status of PML. Additionally, mutations in NFATc3 SUMOylation sites did not affect its binding to PML. The PML-NFATc3 axis regulates Lgr5 and Olfm4 expression, and co-expression of NFATc3 and PML synergistically upregulated these genes. Arsenic sulfide treatment reduced this synergistic effect, indicating its potential as a modulator. This study provides new insights into the regulatory mechanisms of NFATc3 and PML, suggesting potential therapeutic targets in cancer.
{"title":"NFATc3 and PML synergistically regulate tumor-associated gene expression in a SUMOylation-Independent manner","authors":"Ting Kang , Ruizhe Huang , Ruiheng Wang , Han Liu , Siyu Chen","doi":"10.1016/j.biochi.2025.09.002","DOIUrl":"10.1016/j.biochi.2025.09.002","url":null,"abstract":"<div><div>The nuclear factor of activated T cells 3 (NFATc3) plays a significant role in various cancer-related processes, but its interactions with transcriptional modulators, particularly Promyelocytic Leukemia protein (PML), remain poorly understood. PML, a nuclear scaffold protein, is involved in tumor suppression and transcriptional regulation. This study investigates the interaction between NFATc3 and PML, focusing on the role of SUMOylation and its impact on downstream target genes. In vitro experiments, including mass spectrometry and Co-immunoprecipitation (Co-IP), were conducted to explore this interaction. Additionally, constructs with lysine-to-arginine (K→R) mutations at key SUMOylation sites were generated to determine whether PML SUMOylation is necessary for its interaction with NFATc3. We also assessed the impact of NFATc3 SUMOylation on its binding to PML. Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR) were employed to measure the expression of downstream genes (Lgr5 and Olfm4) under NFATc3 and PML overexpression or knockdown conditions. Pharmacological treatment with arsenic sulfide (As<sub>4</sub>S<sub>4</sub>) was used to further investigate modulation of the PML-NFATc3 axis. Our findings revealed that the NFATc3-PML interaction is independent of the SUMOylation status of PML. Additionally, mutations in NFATc3 SUMOylation sites did not affect its binding to PML. The PML-NFATc3 axis regulates Lgr5 and Olfm4 expression, and co-expression of NFATc3 and PML synergistically upregulated these genes. Arsenic sulfide treatment reduced this synergistic effect, indicating its potential as a modulator. This study provides new insights into the regulatory mechanisms of NFATc3 and PML, suggesting potential therapeutic targets in cancer.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 207-217"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145034798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.biochi.2025.09.001
Priscilla Augusta de Sousa Fernandes , Rodrigo Santos Aquino de Araújo , Gabriel Gonçalves Alencar , Sheila Alves Gonçalves , Gildênia Alves de Araújo , Ewerton Yago de Sousa Rodrigues , Daniel Sampaio Alves , Ray Silva de Almeida , Cícera Datiane de Morais Oliveira-Tintino , Maria Gabriella S. Sidrônio , Valnês S. Rodrigues , Emanuelly Karla Araújo Padilha , Edeildo Ferreira da Silva , Anuraj Nayarisseri , Teresinha Gonçalves da Silva , Henrique Douglas de Melo Coutinho , Francisco Jaime Bezerra Mendonça-Junior
In response to the growing threat of antibiotic resistance, this study aimed to identify novel compounds capable of modulating and/or even restoring antibiotic efficacy by inhibiting bacterial efflux pumps. Thirteen 2-aminothiophene (2-AT) derivatives were synthesized and tested against Staphylococcus aureus strains overexpressing NorA and MepA pumps, which confer resistance to fluoroquinolones. Although the 2-ATs displayed little inherent antibacterial activity, several—particularly compounds P4, P7, and P8—significantly potentiated the effects of norfloxacin, ciprofloxacin, and ethidium bromide (EtBr), reducing their Minimum Inhibitory Concentrations (MICs) by up to fourfold. P7 and P8, both 2-aminoselenophene bioisosteres, emerged as especially effective, demonstrating strong efflux pump inhibitory (EPI) activity and, for the first time, confirming their ability to inhibit MepA-mediated efflux in S. aureus. Cytotoxicity assays on VeroE6 and HepG2 cell lines confirmed the safety profile of selected compounds. EtBr accumulation assays and molecular dynamics simulations further supported the mechanism of action, confirming that these derivatives inhibit efflux activity. Overall, the results highlight the potential of 2-AT derivatives—especially P7 and P8—as promising EPIs to combat fluoroquinolone-resistant S. aureus.
{"title":"2-Aminothiophene derivatives reduce resistance to fluoroquinolones in Staphylococcus aureus strains which overexpress NorA and MepA efflux pumps","authors":"Priscilla Augusta de Sousa Fernandes , Rodrigo Santos Aquino de Araújo , Gabriel Gonçalves Alencar , Sheila Alves Gonçalves , Gildênia Alves de Araújo , Ewerton Yago de Sousa Rodrigues , Daniel Sampaio Alves , Ray Silva de Almeida , Cícera Datiane de Morais Oliveira-Tintino , Maria Gabriella S. Sidrônio , Valnês S. Rodrigues , Emanuelly Karla Araújo Padilha , Edeildo Ferreira da Silva , Anuraj Nayarisseri , Teresinha Gonçalves da Silva , Henrique Douglas de Melo Coutinho , Francisco Jaime Bezerra Mendonça-Junior","doi":"10.1016/j.biochi.2025.09.001","DOIUrl":"10.1016/j.biochi.2025.09.001","url":null,"abstract":"<div><div>In response to the growing threat of antibiotic resistance, this study aimed to identify novel compounds capable of modulating and/or even restoring antibiotic efficacy by inhibiting bacterial efflux pumps. Thirteen 2-aminothiophene (2-AT) derivatives were synthesized and tested against <em>Staphylococcus aureus</em> strains overexpressing NorA and MepA pumps, which confer resistance to fluoroquinolones. Although the 2-ATs displayed little inherent antibacterial activity, several—particularly compounds <strong>P4</strong>, <strong>P7</strong>, and <strong>P8</strong>—significantly potentiated the effects of norfloxacin, ciprofloxacin, and ethidium bromide (EtBr), reducing their Minimum Inhibitory Concentrations (MICs) by up to fourfold. <strong>P7</strong> and <strong>P8</strong>, both 2-aminoselenophene bioisosteres, emerged as especially effective, demonstrating strong efflux pump inhibitory (EPI) activity and, for the first time, confirming their ability to inhibit MepA-mediated efflux in <em>S</em>. <em>aureus</em>. Cytotoxicity assays on VeroE6 and HepG2 cell lines confirmed the safety profile of selected compounds. EtBr accumulation assays and molecular dynamics simulations further supported the mechanism of action, confirming that these derivatives inhibit efflux activity. Overall, the results highlight the potential of 2-AT derivatives—especially <strong>P7</strong> and <strong>P8</strong>—as promising EPIs to combat fluoroquinolone-resistant <em>S. aureus</em>.</div></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"239 ","pages":"Pages 191-206"},"PeriodicalIF":3.0,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145126201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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